UDP-beta4-galactosyltransferase (beta4-GT) is a trans-Golgi resident, type II membrane-bound glycoprotein that catalyzes the transfer of galactose to N-acetylglycosamine residues positioned at the non-reducing terminus of glycans forming a beta4-glycosidic linkage. Of the glycosyltransferases well characterized to date, beta4-GT is unique in that its acceptor sugar preference can be modulated in the presence of the "specifier" protein alpha-lactalbumin, a mammalian protein expressed under hormonal control in lactating mammary epithelial cells. Thus, when complexed with alpha-lactalbumin, beta4-GT catalyzes the transfer of galactose to glucose forming the uniquely mammalian disaccharide lactose (Galbeta4-Glu). This proposal takes advantage of previous studies in which we have: (1) Isolated and expressed full-length cDNA clones for bovine, murine and chicken beta4-GT; (2) Developed a panel of species specific McAbs against bovine beta4-GT that recognize discrete polypeptide epitopes including one that overlaps with the alpha-lactalbumin binding site; (3) Demonstrated that an NH2-terminal segment (29 amino acids), containing a truncated cytoplasmic domain and the transmembrane domain of beta4-GT, contains sufficient information for retention of this protein in the Golgi; (4) Isolated and characterized a cDNA clone that encodes a structurally related chicken homologue of beta4-GT. This clone is a new glycosyltransferase; it may potentially encode a reported beta4-GT, distinguished by the absence of a functional alpha-lactalbumin binding domain. Our long range goals are to define in molecular detail, functional domains in beta4-GT including the domain(s) responsible for retention in the trans-Golgi, the catalytic domain and the alpha-lactalbumin binding domain. Toward this end, the Specific Aims of this proposal are: (1) To determine the minimal amino acid requirements for retention of beta4- GT in the trans-Golgi by selective deletion and "domain transfer" experiments. (2) To determine by site-directed mutagenesis the critical amino acid(s) required for substrate binding and alpha-lactalbumin recognition. (3) To determine, using a baculovirus expression vector, if the chicken homologue encodes a form of beta4-GT unresponsive to alpha-lactalbumin or alternatively, a different galactosyltransferase.